My cloned gene in pET23b contains a C-terminal his tag and mature form of protease. Its not getting expressed in e.coli cells.
Even after transformation very little colony forms ~1-5 colonies (only in Rosetta De3 pLysS).
Does anyone have any suggestion of troubleshooting this? I tried with different iptg concentration 0.1mM to 1.0mM. Even added glucose, nothing seems to work. Gel image is attached - Protein molecular weight~51kDa
Lane-1 Marker lane 96Kda, 66Kda, 43 Kda 29.1Kda, 20kda
Lane-2 uninduced cells
Lane-3 to 6 induced with 1mM IPTG at 30min, 60 minute, 90 min, 120 minute
lane-7 blank
lane-8- 180 minutes
lane-9-240 minutes
lane-10- 300 minute
Lane-11-360 Minute
Lane-4
Lane-5
Lane-6
Hi Vishal,
These could be many... issues here.
Change your vector to GST or MBP or sumo or something that is a large protein, not his tag. Since your protein is a mature protease it may be degrading itself. With a large protein as a tag you may see some degraded bands as a starting point. Does this protease has a broad specificity? You can make an active site mutant and see expression. All this is assuming that your protein is expressed and then degraded.
Some things that you can think of:
1. What if your protein is induced without IPTG?
2. Is your plasmid DNA stable in E. coli? IS this the reason you get low number of colonies after transformation?
3. Is your cloned DNA GC or AT rich? or codon bias issues?
4. Some proteins simple cannot be made in E.coli.
5. Structure boundaries in your construct
6. Try varying temperature of induction
You got to try many more expression and vector conditions before you call quits on this one.
Good luck
Hi. In addition to what has been mentioned, it is not clear if you are running lysates or if you have checked the insoluble inclusion body pellet that often forms when the protein of interest aggregates in experiments such as yours. If you have not checked the pellet, you might find your protein of interest is located there.
I think it is difficult to verify the expression of your target protein from the gel. Because when the expression level is low, it is difficult to see the band from unpurified samples. Why don't you check the expression by WB? And as Erik Martin mentioned, protein is often precipitated so I suggest you to check whole cell lysate and supernatant and pellet by anti-His tag antibody..
Yeah, those are some ideas. Here are some others:
Since you only get a few colonies, perhaps sequence your gene in those that come up after transformation. E. coli love to introduce mutations into things that impair their growth.
Try expressing your protein overnight - sometimes it helps. A lot of the things I try to express are insoluble (as Erik mentioned above) so you can also try lowering the temperature during induction - I would suggest 16-18C (as Sujay noted varying temps).
You could try making the strain that normally hosts the protease into the expression vector. This gets around most of the problems with solubility and codon bias. See this paper for a system to do so: One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system. Pubmed URL: http://www.ncbi.nlm.nih.gov/pubmed/17716915
Proteases are very toxic and Lac/T7 system is not a good choice.
Try to use rhamnose or arabinose regulated systems (pBad or pLEMO).
Be also sure that you grow your cells to higher OD600 before induction.
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